The Incidence and Diversity of Plant Viruses in the Tallgrass Prairie Preserve

1. The Incidence and Diversity of Plant Viruses in the Tallgrass Prairie Preserve -Vaskar Thapa, Ulrich Melcher, Daniel McGlinn, Marilyn Roossink, Drew Porter, Rita Marvelli, Tracy Feldman, and Michael Palmer

2. Outline • What is PVBE? • Methods • Field methods • Laboratory methods • Results • Incidence of plant viruses • Diversity of plant viruses • Summary

3. Background: What is PVBE? • Plant Virus Biodiversity and Ecology is one of two scientific theme areas, awarded for research by the National Science Foundation (NSF)-Oklahoma Experimental Program to stimulate Competitive Research (EPSCoR). • Funding provided 2005 to 2008.

4. Objective for PVBE To discover diversity and ecological functions of plant viruses in natural systems.

5. Underlying hypothesisThe distribution and evolution of viruses are determined by complex environmental interactions among many factors including distributions of hosts, vectors, other viruses and climate.

6. Working team • University of Tulsa joining soon • Specializing in • Ecology • Virology • Molecular biology • Genomics • Structural biology and • Bioinformatics.

7. Research site The Nature Conservancy’s Tallgrass Prairie Preserve in Osage County, Oklahoma • Representation of intact native Tallgrass Prairie landscape • 15,000 hectares • more than 700 plant species • 12 vegetation types Palmer, M. W., P. Earls, and J. R. Arévalo. 2000. The vegetation of the Tallgrass Prairie Preserve (unpublished report).

8. Focus of this presentation  Preliminary results from plants collected since May 2005 Analysis of double-stranded (ds) RNA from the plant collected

9. Methods • Field methods • Plants for voucher herbarium • Sample collection for dsRNA isolation • Plant samples for intensive study • Laboratory methods • ds RNA isolation

10. Field methods • Plant sample for voucher herbarium • Sample from each species • Collection irrespective of • symptoms • Collection from sites with • abundance of target species • Record of GPS location • Habitat and individual plant photos • Two repositories for herbarium –OSU and TGPP

11. Field methods .. • Sample collection for ds RNA isolation • 10 grams of young leaves • Transported to the laboratory in a container • with ice packs • Stored in cold room at 4 ْ C before processing • for dsRNA isolation

12. Field methods .. Plant samples for intensive study • Six of the most frequent • plants in tallgrass prairie • vegetation • Represent major taxonomic • groups • Multiple samples from 20 • random plots

13. Ambrosia psilostachya, Cuman ragweed

14. Asclepias viridis, green antelopehorn

15. Panicum virgatum, switchgrass

16. Sorghastrum nutans, Indiangrass

17. Ruellia humilis, fringeleaf wild petunia

18. Vernonia baldwini, baldwin's ironweed

19. Laboratory method for double-stranded RNA isolation Mix vigorously to form emulsion Young leaves (5 g) Centrifuge Grind in liquid nitrogen Transfer top phase into new tube Transfer into 50 ml tube containing 10 ml extraction buffer and 10 mlPh:Ch Final aqueous phase Repeat Ph:Ch extraction Centrifuge to pellet dsRNA Add absolute proof ethanol (16,5% of aqueous volume) Add elution buffer Decant and resuspend in 0,1 mM EDTA / 0.3 M NaOAC Wash in 6 time with application buffer Pass through enocolumn containing CF11powder cellulose binding dsRNA (if ethanol concentration is 16,5%) Precipitate with NaOAc and EtOH overnight at -20ْ C Total NA (for bar coding and making hybridization target) Transfer eluate to a 15 ml tube 1 2 3 4 Lad 1 kb Buffers Transfer to a microcentrifuge tube and fill with cold ethanol to reprecipitate Extraction buffer: 0.1 M NaCl 50 mV Tris, pH 8 1 mV EDNA, pH 8 1% SDS Application buffer: 0.1 M NaCl 50 mM Tris, pH8 0.5 mV EDTA, pH8 16.5% Ethanol Resuspend in50 mkl 0.1 mM EDTA bp Vernonia baldwinii (line 1) and Flavoparmelia sp. (line 4) have no dsRNA. Ambrosia psilostachya (line 2) and Parmelia sp1. (line 3) show bands for dsRNA Elution buffer: 0.1 M NaCl 50 mV EDTA, pH 8 12,2 2 1,6 Check the dsRNA by electrophoresis on a 1.5% agarose gel in 0.5X TBE II 1 506,5 396 344 298 Protocol for ds RNA isolation adopted from M. Roossinck, 2005

20. Results • 635 specimen from 485 species, 307 genera and 91 families collected. • 592 specimens analyzed for • ds RNA • gels of 592 these specimens • 308 of the are putatively • positive for dsRNA (i.e. • probable viruses)

21. Distribution within the top plant families 2 = 37.39, p = 0.00

22. Double-stranded RNA in native and exotic species 2 = 0.06, p = 0.8

23. Distribution of dsRNA in different life forms 2 = 1.23, p = 0.87

24. Distribution of dsRNA in different taxonomic groups 2 = 13.81, p = 0.00

25. Distribution of dsRNA in six selected species 2 = 9.76, p = 0.08

26. Viral Diversity • Tooearly to comment on plant • virus diversity in TGPP • Gel analysis shows wide • variation in banding patterns • Different banding patterns within • and across species.

27. Caveats • The results are preliminary, based on a limited • sample • dsRNA is not unique for plant virus, it may be from • fungal or arthropod viruses • Viruses of low titer may have been missed • DNA viruses are not assessed. • The reading of the gels has some subjectivity; this • will be resolved in the sequencing phase of PVBE

28. Conclusions • 50% of plant samples contain dsRNA, indicating viruses are widespread in nature. • Viruses are frequent in all growth forms, life histories, and taxonomic groups.

29. Acknowledgments • Following persons who help us in plant collection • Pete Earls Ray Moranz • Fumiko Shirakura Josh Lofton • M.Hara Mari Carmen Cobo • Will Lowry Laxman Karki • Shyam Thomas Katie Lewis • Rest of all team members of PVBE